Prediction and Prognosis for Immunotherapy of Intra-tumoral Interleukins Expression Patterns in Non-small Cell Lung Cancer / 中山大学学报(医学科学版)

ObjectiveTo identify the relationship between tumor tissue interleukins （ILs） and non-small cell lung cancer （NSCLC） patients with poor response to immune checkpoint blockade （ICB） therapy， and to investigate the differential expression of ILs in tumor of NSCLC patients as well as its effect on ICB response and prognosis. MethodsA total of 61 patients diagnosed with NSCLC and treated with ICB were retrospectively collected from the data of a previous study. We obtained transcriptome sequencing data from tumor tissues and survival data of the patients before ICB treatment. Using bioinformatics methods， we screened for ILs that significantly affected the efficacy and prognosis of ICB treatment. We evaluated the efficacy of ICB treatment using progressive-free survival （PFS） and assessed the prognosis using overall survival （OS）. The Kaplan-Meier survival curve and ROC curve were used to analyze the predictive effect and efficacy of ILs on the efficacy and prognosis of ICB in NSCLC patients. ResultsThe results of the univariate Cox regression analysis in our study showed that nine ILs were found to be associated with OS of NSCLC patients treated with ICB at a significance level of P < 0.1. Further multivariate analysis revealed that high expression of IL-11， IL-17D， and IL-36A was significantly associated with poor prognosis in these patients （P < 0.05）. The results from the Kaplan-Meier survival curve analysis revealed a significant negative correlation between the high expression of IL-17D and both PFS and OS in NSCLC patients. Specifically， patients with IL-17D high expression had a median PFS of 3.1 months compared with 6.5 months in low expression patients ［95% confidence interval （CI） （1.178， 3.655）， P = 0.009］. Similarly， the median OS was 9.8 months in the high expression group versus 21.8 months in the low expression group ［95%CI （1.116， 4.392）， P = 0.018］. ROC curve showed that the prediction performance was favorable ［AUCPFS = 0.702，95%CI （0.562， 0.842）， P = 0.027； AUCOS = 0.684， 95%CI （0.550， 0.818）， P = 0.014］. Although IL-11 and IL-36A alone were not significant predictors of PFS and OS in NSCLC patients， the median PFS and OS were notably shortened to 2.2 months （P = 0.003） and 3.0 months （P < 0.001）， respectively， when high expression of IL-11 and IL-36A was combined with high expression of IL-17D. The ROC curve analysis demonstrated an improvement in prediction efficiency for both PFS and OS in NSCLC patients ［AUCPFS = 0.748， 95%CI （0.615， 0.880）， P = 0.007； AUCOS = 0.703， 95%CI （0.573， 0.833）， P = 0.007］. ConclusionThe results suggest that high expression of IL-11， IL-17D， and IL-36A is associated with a higher risk of disease progression which correlates to poor PFS and OS in NSCLC patients.

An Overview and Interpretation of Important Clinical Studies on Non-small Cell Lung Cancer in 2021 / 中国肺癌杂志

Though the coronavirus disease is still raging in 2021, clinical research on non-small cell lung cancer (NSCLC) did not stop. However, benefiting from advances in lung cancer treatment modality, NSCLC patients have experienced significant improvements in overall survival and quality of life. Currently, research advances on targeted therapy and immunotherapy have together transformed the status of postoperative adjuvant therapy and established a new standard treatment modality for resectable NSCLC. There are equally important research advances in locally advanced and advanced NSCLC, including new treatment modalities, new therapeutic agents, etc., all of which bringing more options for clinical treatment. These therapies will bring changes to NSCLC and will gradually lead to the chronicity of lung cancer in the foreseeable future. Therefore, this paper reviews important studies that will change clinical practice in NSCLC treatment and noteworthy research advances in 2021.
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A Chinese host genetic study discovered type I interferons and causality of cholesterol levels and WBC counts on COVID-19 severity

As of early May 2021, the ongoing pandemic COVID-19 has caused over 160 million of infections and over 3 million deaths worldwide. Many risk factors, such as age, gender, and comorbidities, have been studied to explain the variable symptoms of infected patients. However, these effects may not fully account for the diversity in disease severity. Here, we present a comprehensive analysis of a broad range of patients laboratory and clinical assessments to investigate the genetic contributions to COVID-19 severity. By performing GWAS analysis, we discovered several concrete associations for laboratory features. Based on these findings, we performed Mendelian randomization (MR) analysis to investigate the causality of laboratory traits on disease severity. From the MR study, we identified two causal traits, cholesterol levels and WBC counts. The functional gene related to cholesterol levels is ApoE and people with particular ApoE genotype are more likely to have higher cholesterol levels, facilitating the process that SARS-CoV-2 binds on its receptor ACE2 and aggravating COVID-19 disease. The functional gene related to WBC counts is MHC system that plays a central role in the immune system. The host immune response to the SARS-CoV-2 infection greatly affects the patients severity status and clinical outcome. Additionally, our gene-based and GSEA analysis revealed interferon pathways, including type I interferon receptor binding, regulation of IFNA signaling, and SARS coronavirus and innate immunity. We hope that our work will make a contribution in studying the genetic mechanisms of disease illness and serve as useful reference for the clinical diagnosis and treatment of COVID-19.

Genomic epidemiology of SARS-CoV-2 in the United Arab Emirates reveals novel virus mutation, patterns of co-infection and tissue specific host responses

To unravel the source of SARS-CoV-2 introduction and the pattern of its spreading and evolution in the United Arab Emirates, we conducted meta-transcriptome sequencing of 1,067 nasopharyngeal swab samples collected between May 9th and Jun 29th, 2020 during the first peak of the local COVID-19 epidemic. We identified global clade distribution and eleven novel genetic variants that were almost absent in the rest of the world defined five subclades specific to the UAE viral population. Cross-settlement human-to-human transmission was related to the local business activity. Perhaps surprisingly, at least 5% of the population were co-infected by SARS-CoV-2 of multiple clades within the same host. We also discovered an enrichment of cytosine-to-uracil mutation among the viral population collected from the nasopharynx, that is different from the adenosine-to-inosine change previously reported in the bronchoalveolar lavage fluid samples and a previously unidentified upregulation of APOBEC4 expression in nasopharynx among infected patients, indicating the innate immune host response mediated by ADAR and APOBEC gene families could be tissue-specific. The genomic epidemiological and molecular biological knowledge reported here provides new insights for the SARS-CoV-2 evolution and transmission and points out future direction on host-pathogen interaction investigation.

Initial Study of Human Genetic Contribution to COVID-19 Severity and Susceptibility

The COVID-19 pandemic has accounted for more than five million infections and hundreds of thousand deaths worldwide in the past six months. The patients demonstrate a great diversity in clinical and laboratory manifestations and disease severity. Nonetheless, little is known about the host genetic contribution to the observed inter-individual phenotypic variability. Here, we report the first host genetic study in China by deeply sequencing and analyzing 332 COVID-19 patients categorized by varying levels of severity from the Shenzhen Third Peoples Hospital. Upon a total of 22.2 million genetic variants, we conducted both single-variant and gene-based association tests among five severity groups including asymptomatic, mild, moderate, severe and critical ill patients after the correction of potential confounding factors. The most significant gene locus associated with severity is located in TMEM189-UBE2V1 involved in the IL-1 signaling pathway. The p.Val197Met missense variant that affects the stability of the TMPRSS2 protein displays a decreasing allele frequency among the severe patients compared to the mild and the general population. We also identified that the HLA-A*11:01, B*51:01 and C*14:02 alleles significantly predispose the worst outcome of the patients. This initial study of Chinese patients provides a comprehensive view of the genetic difference among the COVID-19 patient groups and highlighted genes and variants that may help guide targeted efforts in containing the outbreak. Limitations and advantages of the study were also reviewed to guide future international efforts on elucidating the genetic architecture of host-pathogen interaction for COVID-19 and other infectious and complex diseases.

Stability of SARS-CoV-2 on environmental surfaces and in human excreta

At room temperature, SARS-CoV-2 was stable on environmental surfaces and remained viable up to 7 days on smooth surfaces. This virus could survive for several hours in feces and 3-4 days in urine.

Functions of lamin B1 and the new progress of its roles in neurological diseases and tumors / 生物工程学报

Lamin B1 is one of the essential members of the nuclear lamina protein family. Its main function is to maintain the integrity of nuclear skeleton, as well as to participate in the cell proliferation and aging by affecting the chromosome distribution. gene expression, and DNA damage repair. The abnormal expression of lamin B1 is related to certain diseases, including neurological diseases [e.g. neural tube defects (NDTs), adult-onset autosomal dominant leukodystrophy (ADLD)] and tumors (e.g. pancreatic cancer). It is also a potential tumor marker as well as drug target. Further research on lamin B1 will help people understand the molecular mechanism of the emergence and development of neural system diseases and tumors, and define a new future in drug target.

Study on expression of RACK1,Src and Bcl-2 protein and their correlation in gastric carcinoma tissues and adjacent carcinomatous tissues / 重庆医学

Objective To investigate the expression change of RACK1 ,Src and Bcl‐2 in gastric carcinoma tissue and adjacent carcinomatous tissue .Methods Eighty specimens of gastric carcinoma and adjacent carcinomatous tissues in our hospital from Au‐gust 1 ,2011 to February 1 ,2014 were collected .The immunohistochemistry staining and Western blotting methods were adopted to detect the expression of RACK1 ,Src and Bcl‐2 protein in gastric carcinoma and adjacent carcinomatous tissues ,and their correlation was analyzed and performed the statistical analysis by combining with the clinicopathological data .Results The immunohistochem‐istry staining and Western blotting detection displayed that the expression positive rate and expression level of RACK 1 in gastric carcinoma tissue were obviously lower than those in the adjacent carcinomatous tissue ,while the expression positive rate and ex‐pression level of Src and Bcl‐2 in gastric carcinoma tissue were obviously higher than those in the adjacent carcinomatous tissue ,the differences were statistically significant (P0 .05) .Conclusion The expression of RACK1 in gastric carcinoma tissue is significantly decreased ,while the expres‐sions of Src and Bcl‐2 are increased .

VEGF and VEGFR Expressions in Non-small Cell Lung Cancer with Malignant Pleural Effusion / 中国医科大学学报

Objective To investigate the expression of VEGF and VEGFR in non?small cell lung cancer patients with malignant pleural effusion, and analyze the relationship between VEGF and VEGFR and malignant pleural effusion. Methods The VEGF and VEGFR expression were detect?ed using immunohistochemistry in pleural and lung tumors tissues of 30Ⅳstage non?small cell lung cancer patients,and the relationship between VEGF and VEGFR expression and malignant pleural effusion was analyzed. Results The expression of VEGF and VEGFR in pleural tissue of pa?tients with malignant pleural effusion was significantly higher than those without malignant pleural effusion(P<0.05). Conclusion There is a very close correlation between high expression of VEGF and VEGFR and formation of malignant pleural effusion.

Analysis on gene sequence of HIV isolated from men who have sex with men in Beijing / 中华流行病学杂志

Objective To analyze genetic characteristics of HIV isolated from men who have sex with men(MSM) in Beijing and predict the epidemic trend in this population.Methods All of the HIV gene sequences in our laboratory obtained from MSM in Beijing were used,which were aligned with all of the HIV gene sequences from MSM and other populations in China downloaded from Los Alamos HIV Database.Phylogenetic trees were constructed by using software PhyML 3.0,based on which the relationships of prevalent HIV strains between Beijing MSM and other populations in China were further explored.The evolution rate,the time of most recent common ancestor (tMRCA),the epidemic parameters,the reproductive number (R0) were calculated by using software BEAST to predict HIV evolution and epidemic characteristics.Results Multiple HIV subtypes,including subtype B,CRF01_AE and CRF07_BC,were found to be prevalent among MSM in Beijing.In ML tree constructed based on strains from the whole country,three clusters including B-1,CRF01_AE-1,and CRF01_AE-2 were found among the MSM in Beijing (accounting for 40％).At least three independent introduction of B 1 cluster strains into Beijing MSM were found,which were at March 1991 (July 1984-February 1997),January 1994 (January 1989-January 1998),April 1991 (August 1984-January 1996).For CRF01_AE strains,two clusters including CRF01_AE-1 and CRF01_AE-2 were introduced into the population at December 2000 (March 1998-January 2003) and December 2001 (January 2000-July 2003) respectively.The population epidemiology of HIV in Beijing MSM was reconstructed based on sequences.The CRF01_AE-1 cluster spread more quickly than the other two clusters,and the evolution rate was higher.Conclusion Multiple HIV subtypes were found prevalent among MSM in Beijing.Although subtype B strain was introduced into Beijing MSM earlier than CRF01_AE strain,CRF01_AE strain increased more quickly than subtype B strain.More research and control of the CRF01_AE prevalence will be helpful for prevention and control of HIV epidemic in MSM in Beijing.

Similar Neutralizing Activity in the HIV-1 Infected Long Term Non-progressors(LTNPs) and Typical Progressors(TPs) / 中国病毒学·英文版

Neutralizing antibodies are considered to be an important protective parameter used in HIV-l vaccine evaluation.However,the exact role that neutralizing antibodies plays in controlling the disease progression of HIV-1 infected peoples is still undetermined.In this paper,we compared the protective function of the neutralizing antibody response in the plasma from LTNP and TP against clade B and clade C pseudoviruses.No difference in the neutralizing activities between the plasma from LTNP and TP was found,which was consistent with the most recent reports.In addition,no correlations between the titer or breadth and CD4+ or viral load in HIV-1 infected individuals were found.The protective roles played by neutralizing antibodies in controlling disease progression of HIV-1 infected people need to be considered in a new viewpoint.

Evaluation of an in-house method for HIV-1 drug resistance genotyping test / 中华检验医学杂志

ObjectiveTo evaluate the sensitivity and accuracy of an in-house detecting method of HIV-1 genotypic drug resistance system. MethodsTotally 130 serum specimens from Henan and Guangxi province were collected from April 2004 to October 2008 and tested in the Military HIV Testing Center of China. ViroSeqTM v2.0 (Abbott, Switzerland), a US FDA approved HIV genotypic drug resistance detecting system was utilized as the reference method. All the specimens were detected by the novel in-house method and the reference method to validate the difference in amplifying efficiency, drug resistance mutation detection and drug resistance report. ResultsConcerning the 14 850 known drug resistance mutation sites,14 752 (99. 3％ ) mutations can be detected by both of the two methods. Rates of concordance of detection in the regions of protease inhibitors-, reverse transcriptase inhibitors- and both two classes inhibitors-resistance were99.7％ ( Kappa =0. 909 9 , P ＜0. 01 ) , 99. 0％ (Kappa=0.952 1, P＜0. 01) and99.3％ (Kappa=0. 948 8, P ＜ 0. 01 ) respectively. Drug resistance reports from these two systems showed similar results (Kappa = 0. 637 4, P ＜ 0. 01 ). The in-house detecting system identified 34 novel mutations besides the ViroSeqTM drug resistance mutation database ( ViroSeqTM software v2. 7). Two mutations, V179F and K238T,had significant effect on HIV drug resistance. ConclusionsThe in-house genotyping system is an accurate,cost-effective method and has a high concordance with commercial ViroSeqTM genotyping system. Database from the in-house assay was superior to this of the ViroSeqTM assay.

Study on the evolution of the minor resistant mutations and the primary resistance in rural areas of Henan / 中华微生物学和免疫学杂志

Objective To evaluate the antiretroviral therapy(ART),analyze the prevalence of resistance in rural areas,Henan,and explore the presence of minor resistant variants in pre-ART.Methods One hundred and forty-nine AIDS patients initiating ART were recruited and investigated at intervals of 6 months. Method of In-house developed by our laboratory for genotypjc resistance test was to analyze the occurrence of resistance among the failure of ART,and the allele-specific real.time PCR(ASPCR)was used to detect the minor resistant variants at the baseline samples once the resistance occurred.Results Vimlload significantly decreased among the patients who received ART(t=275,P=0.0001),but the absolute counts of CD4+T lymphocytes had no significant change(t=1.765 168,P=0.0852).Rate of resistance among the patients of treatment failure was 4.88%.The result of ASPCR in the survey of baseline showed that the minor resistant variants of M184V were detected in 7 patients and mutation K103N presented in 5 patients.Conclusion The primary drug-resistant straias in the untreated patients were found in Henan,and they might develop the dominant resistance strains and bring about the failure of ART.

Effect of Morphine on the replication of HIV-1 in MT2 and macrophage / 中华检验医学杂志

Objective To determine whether Morphine has the ability to enhance HIV-1 replication in MT2 and Macrophage in vitro and assess the influence of Naloxone on Morphine2s effect.Methods MT2 cells were randomly assigned into 4 groups: (1) Morphine treatment for MT2 group, (2) Morphine+Naloxone co-treatment for MT2 group, (3) Naloxone treatment for MT2 group and (4) MT2 Control;Macrophages were also randomly assigned into 4 groups: (5) Morphine treatment for Macrophage group, (6) Morphine+Naloxone co-treatment for Macrophage group, (7) Naloxone treatment for Macrophage group and (8) Macrophage Control. Group (2), (3), (6) and (7) were pre-treated with 10-8 mol/L Naloxone for 0.5 h, and then group (1) and (2) were treated with 10-12, 10-10 and 10-8 mol/L Morphine for 24 h;group (5) and (6) were disposed of 10-10 mol/L Morphine for 24 h.All 8 groups were added in HIV-1 viral strain with 50% tissue culture infective dose(TCID50).P24 antigen in MT2 cells culture supernatant at day 3, 4, 5 and 6, and in Macrophages culture supernatant at day 4, 6, 8, 10 and 12 after infection were determined with ELISA.Student2s t-test and ANOVA were used to compare the differential expression in different groups, and repeated measures ANOVA was used to compare the increasing or decreasing expression of p24 antigen in morphine treatment groups than that in the control group at different time points.Results On the 3rd day of infection with HIV-1 in MT2 cells, the expression of p24 antigen in 10-12, 10-10 and 10-8mol/L dose of group (1) were (4.44?.30), (5.59?.25) and (4.60?.24) ng/ml respectively, compared to control[(1.93?.05) ng/ml, t= 14.15, 24.74 and 19.14, all P<0.01].On the 4th day, 10-12, 10-10 and 10-8mol/L dose of group (1) resulted in a significant increase of p24 antigen expression [(24.30?.66), (31.73?.17) and (26.02?.37) ng/ml]in culture supernatants compared to control[(8.03?.09) ng/ml, t=10.59, 34.92 and 81.2, all P<0.01].On the 5th day, the expression of p24 antigen in 10-12, 10-10 and 10-8 mol/L dose of group (1) were (56.30?.26), (81.77?.49) and (63.66?.57) ng/ml respectively, compared to control [(15.30?.91) ng/ml, t= 45.83, 43.51 and 30.07, all P<0.01].On the 6th day, the expression of p24 antigen in 10-12, 10-10 and 10-8 mol/L dose of group (1) were (150.70?.97), (243.09?.93) and (173.72?.73) ng/ml respectively, compared to control [(41.01?.84) ng/ml, t= 21.09, 39.02 and 29.55, all P<0.01].The enhanced multiple of p24 antigen expression in three doses of morphine treatment group compared to control increased with HIV-1 infected MT2 cells time, trend analysis of repeated measurements showed statistically significant time effect (F=842.18, P<0.01). On the 4th day of infection with HIV-1 in Macrophage cells, the expression of p24 antigen in 10-12, 10-10 and 10-8 mol/L dose of group (5) were (0.68?.15), (0.87?.41) and (0.75?.09) ng/ml respectively, compared to control [(0.60?.01) ng/ml, t= 7.27, 11.06 and 3.02, all P<0.05]. On the 6th day, 10-12, 10-10 and 10-8 mol/L dose of group (5) resulted in a significant increase of p24 antigen expression[(1.64?.57) , (2.07?.12 ) and (1.75?.17) ng/ml]in culture supernatants compared to control [(1.16?.07) ng/ml, t=8.93, 11.3 and 5.45, all P<0.01].On the 8th day, the expression of p24 antigen in 10-12, 10-10 and 10-8 mol/L dose of group (5) were (6.31?.17), (8.81?.34) and (7.19?.11) ng/ml respectively, compared to control [(3.84?.45) ng/ml, t=8.83, 15.11 and 12.42, all P<0.01]. On the 10th day, the expression of p24 antigen in 10-12, 10-10 and 10-8 mol/L dose of Morphine treated group were (32.30?7.55), (50.74?7.55) and (39.74?.56) ng/ml respectively, compared to control [(17.55?.86) ng/ml, t= 13.65, 17.84 and 36.69, all P<0.01].The enhanced multiple of p24 antigen expression in three doses of group (5) compared to control increased with HIV-1 infected Macrophage cells time, trend analysis of repeated measurements showed statistically significant time effect (F=135.58, P<0.01).Conclusions Morphine has the ability to enhance HIV-1 replication in MT2 cell and Macrophage. This Morphine-mediated increase of p24 antigen expression can be blocked by Naloxone.

Mechanisms of bone metastasis in prostate cancer / 国际肿瘤学杂志

The molecular and cellular mechanisms of bone metastasis in prostate cancer remain unclear. Current researches focus on chemotaxis of tumor cells in bone metastasis, interactions between tumor cells and bone micro-environment as well as the vicious circle among tumor cells, osteoclasts, osteoblasts and bone matrix.

Evolvement of HIV-1 drug resistant mutations in vitro without drug pressure / 中华微生物学和免疫学杂志

Objective To isolate stable passage primary HIV-1 drug resistant strains and observe replication dynamics of the drug resistant isolates and evolvement tendency of the drug resistant mutations in vitro.Methods Peripheral blood mononuclear cells(PBMCs)from 15 AIDS patients receiving highly active antiretroviral therapy(HAART)were collected,and the primary HIV-1 stains were separated utilizing co-cultivated with PBMCs from normal people.HIV-1 pol genes from those strains were obtained by RT-PCR and sequenced.The drug resistant mutations were analyzed in the Stanford HIV Drug Resistance Database.Results Eight strong positive strains were isolated from 15 AIDS patients with viral loads higher than 1000 copies/ml,and two of them were drug resistant.Drug resistant mutations of the two strains were respectively K103N/K238T and M184V/K103N/Y181C/H221Y which show high-level resistance to NVP and 3TC/NVP,respectively.K103N,M184V,Y181C and H221Y exist stably in the environment without drug pressure,however,RT K238T reverted to K238.Conclusion Two drug resistant strains were successfully isolated in vitro without drug pressure.Strains with K103N shows superior fitness and can exist steadily.Strains with M184V and K103N/Y181C/H221Y can also replicate stably in vitro without drug pressure.NNRTI mutation K238T reproduces astatically,which suggests that RT 238 codon might revert gradually to wild genotype.

Indinavir Resistance Evolution in One Human Immunodeficiency Virus Type 1 Infected Patient Revealed by Single-Genome Amplification / 中国病毒学·英文版

Human Immunodeficiency Virus Type 1 exists in vivo as quasispecies, and one of the genome's characteristics is its diversity. During the antiretroviral therapy, drug resistance is the main obstacle to effective viral prevention. Understanding the molecular evolution process is fundamental to analyze the mechanism of drug resistance and develop a strategy to minimize resistance. Objective: The molecular evolution of drug resistance of one patient who had received reverse transcriptase inhibitors for a long time and had treatment which replaced Nevirapine with Indinavir was analyzed, with the aim of observing the drug resistance evolution pathway. Methods: The patient, XLF, was followed-up for six successive times. The viral populations were amplified and sequenced by single-genome amplification. All the sequences were submitted to the Stanford HIV Drug Resistance Database for the analysis of genotypic drug resistance. Results: 149 entire protease and 171 entire reverse transcriptase sequences were obtained from these samples, and all sequences were identified as subtype B. Before the patient received Indinavir, the viral population only had some polymorphisms in the protease sequences. After the patient began Indinavir treatment, the variants carrying polymorphisms declined while variants carrying the secondary mutation G73S gained the advantage. As therapy was prolonged, G73S was combined with M46I/L90M to form a resistance pattern M46I/G73S/L90M, which then became the dominant population. 97.9% of variants had the M46I/G73S/L90M pattern at XLF6. During the emergence of protease inhibitors resistance, reverse transcriptase inhibitors resistance maintained high levels. Conclusion: Indinavir- resistance evolution was observed by single-genome amplification. During the course of changing the regimen to incorporate Indinavir, the G73S mutation occurred and was combined with M46I/L90M.

In vitro selection and identification of HIV strain which is resistance to two new HIV-1 nonnucleoside reverse transcriptase inhibitors / 药学学报

JB25 and JB26 are new HIV-1 nonnucleoside reverse transcriptase inhibitors, and show potent anti-HIV activities. Sequential passage experiments with wild-type virus were performed to select and identify mutations induced by these two compounds in vitro. For the initial passage, compounds were present at approximately 2-fold IC50 in MT-2 cells. When cytopathic effect (CPE) was observed in more than 75% of the cells, the culture supernatants were collected. For the subsequent passages, fresh MT-2 cells were infected with 1 mL supernatants from the previous passage (regardless of the virus titer) and cultured in the presence of the compounds at concentrations that were increased 2-fold compared with that in the previous passage. This procedure was repeated with increasing concentrations for 12 passages. JB25 had amino acid substitution L100I (TTA-->ATA) at passage 6, and then changed into 100 M (ATA-->ATG) at passage 12, which was rare mutation form and had not been reported. At the same time, Y188C (TAT-->TGT) mutation appeared at passage 10. For JB26, there was a L100I (TTA-->ATA) mutation at passage 10. In a word, JB25 and JB26 showed a low genetic barrier to the development of resistance, and the resistance to JB26 developed slower than JB25. The mutations selected by JB25 and JB26 were mainly associated with codons 188 and 100 of HIV-1 reverse transcriptase.

Allele-specific real-time PCR for the detection of minor HIV-1 variants / 中华微生物学和免疫学杂志

Objective To develop and evaluate the allele-specific real-time PCR(ASPCR) assay for the detection of minor HIV-1 variants. Methods We developed and evaluated the ASPCR assay, using the K103N mutation site as a model system. We constructed plasmids as standards and designed specific and non-specific primers to discriminate the wild-type and mutant plasmids in the real-time PCR using SYBR green as fluorescence reporter. And then we evaluated the sensitivity, accuracy, reproducibility of ASPCR assay and detected the control samples. Results The specific primer can discriminate the wild-type and mutant plasmids including resistant mutation successfully. The sensitivity of ASPCR assay can achieve less than 0.01% and the accuracy of this method is down to 0.1%. The Intra-assay coefficient of variation is less than 0.7 and the Inter-assay coefficient of variation is less than 1.6. Conclusion ASPCR is a sensitive, accurate and rapid method to detect the minor HIV-1 variants which have resistant mutations and it can be used widely in HIV research. ASPCR also can provide earlier and more resistant information to the clinical therapy.

Correction of short collumella by using the free composite graft of ear lobe / 中华医学美学美容杂志

Objective To investigate a simple and safe method for correction of the flat nose tip with the short columella. Methods The flat nose tip with the extend strut graft was corrected by using the rib cartilage or Medpor and the secondary defect of columella was repaired with the free composite graft of ear lobe in 8 cases of rhinoplasties. Results The free composite graft of ear lobe survived well and the color was similar to the neighboring tissue. There was no obviously secondary deformation at the donor site in all the cases by following-up from 1 to 2 years. Conclusions It is a simple and effective method without secondary deformation to repair the short columella by using the free composite graft of ear lobe in rhinoplasty.